Influenza viruses exploit sophisticated host cell machinery to replicate, causing both seasonal epidemics and unpredictable pandemics. Studying the host cellular factors interacting with conserved domains of viral proteins will help us to identify key host proteins for the virus infection. This will not only strengthen our understanding of the precise mechanisms of the virus life cycle, but also pave new avenues for anti-viral development. The cytoplasmic tail of M2 ion channel (M2/CT) is one of these highly conserved domains. It is fully accessible to the host cell machinery after fusion of the virus envelope with the endosomal membrane and during the trafficking, assembly, and budding processes. I hypothesized that recruitment of host cellular factors by M2/CT may regulate the M2-dependent stages of the virus life cycle. Through a large scale yeast two-hybrid (Y2H) screen with the M2/CT used as bait, the human annexin A6 was identified as a novel host cell interactant and this interaction was further confirmed by both GST pull-down assay on purified proteins and co-immunoprecipitation assay on virus infected cells. A functional characterization of this novel interaction demonstrated that depletion of annexin A6 could enhance the virus production, while its overexpression could reduce the virus propagation, which indicates that annexin A6 is a negative regulator of the virus infection. However, I found that the virus infection could not induce any changes of annexin A6 expression. Therefore, the annexin A6-mediated regulation may depend on the subcellular localization where the interaction with M2/CT occurs. To decipher which step of the virus replication is regulated, we dissected the virus life cycle and found that modulation of annexin A6 expression had no effect on the early stages of the virus life cycle or on viral RNA replication but impaired the release of progeny virus, as suggested by delayed or defective budding events observed at the plasma membrane of virus-infected and annexin A6-overexpressing cells during a transmission electron microscopy study. To further decipher the underlying molecular mechanisms, the contribution of annexin A6-mediated plasma membrane lipid rafts reorganization through cholesterol homeostasis modulation and cortical actin cytoskeleton remodeling was also investigated. In conclusion, here I have identified the human annexin A6 as a novel host cell interactant of M2/CT that negatively modulate the influenza virus infection by impairing the virus budding and release. This work further supports the idea that M2 is a multifunctional protein and is also consistent with the discovery by Rossman et al. that M2/CT mediates the virus budding process (Rossman et al., 2010). This study further emphasizes the importance of host cell interactants of M2/CT in this process. Regarding the biology of annexins, this study also adds a new member of this protein family in the list of regulators of influenza virus infection. / published_or_final_version / Public Health / Doctoral / Doctor of Philosophy

Introduction With the emergence of the H5N1 virus in humans that was of entirely avian origin, a better understanding of potential receptors of the influenza A virus is needed. It is widely accepted that terminally sialylated glycoconjugates on the surface of the red blood cells are receptors to which the influenza A virus binds to and causes the agglutination of red blood cells. By isolating glycans found on red blood cells, perhaps it is possible to find potential receptors that influenza A virus has preferential binding to. There has been a general shift of using turkey red blood cells rather than chicken red blood cells for the haemagglutination of viruses over the past few decades. The influenza virus’s loss in ability to agglutinate chicken red blood cells but keep the ability to agglutinate turkey red blood cells a puzzling mystery. By comparing the differences, perhaps it is possible to elucidate structures influenza viruses prefer to bind to. Methods Mass spectrometricanalysis of purified glycans will allow us to narrow down the structures of the most abundant glycans found on the erythrocyte surface. Lectin staining with flow cytometry is used to identify the receptor specificity of the influenza viruses. Haemagglutination assay in conjunction with glycan binding array data from the CFG will allow us to pinpoint the possible structures that give the viruses the ability to bind using treatments with sialidases. Results The mass spectrometric data was good and the basic glycan structures were elucidated according to their mass to charge ratio. The proportion of the different glycans for each of the erythrocyte type was clearly shown. The lectin staining gave more accurate results have selecting a single cell population and it was clear that turkey red blood cells had more α2,3 and α2,6 linked glycans than the chicken red blood cells. The haemagglutination assay aided the identification of differentiating theα2,3 linked and α2,6 linked liking viruses. The glycan array binding data was obtained but some results were absent. Conclusion It is certain that turkey red blood cells are better than chicken red blood cells for use in the haemagglutination assays as they present some glycans that are present in human bronchial epithelials. The most abundant sialylated glycan are the α2,6 linked sialylated glycans. The most abundant glycans on chicken red blood cells are either absent or in low abundance on the HBEs. Though the turkey red blood cells have some glycans that are present on HBEs, the glycan profile is very different. To agglutinate red blood cells, viruses are likely to bind to the bisecting glycans and shorter antennae glycans. In methodology: for lectin staining experiments, one must take caution in the lectin used as it may affect results. Sialidase S treated RBCs are a good method to distinguish α2,3 linked glycans liking viruses. / published_or_final_version / Pathology / Master / Master of Medical Sciences

Neuraminidase (NA) inhibitors are one of two classes of antiviral compounds available for the control of influenza infections. The H275Y NA mutation confers resistance to the NA inhibitor oseltamivir carboxylate among the N1 influenza subtype and has been identified from resistant variants with distinct epidemiological outcomes in human. Specifically, dominance of oseltamivir-resistant variant of the A/Brisbane/59/2007-like viruses was reported during the 2008 to 2009 influenza season, until it was replaced by the 2009 pandemic H1N1 virus [A(H1N1)pdm09]. Since the emergence of the 2009 pandemic, the fitness and transmission potential of oseltamivir-resistant A(H1N1)pdm09 variants carrying the H275Y mutation has been a concern. This project aims to systematically evaluate the fitness of viruses carrying the H275Y mutation for A(H1N1)pdm09 and seasonal H1N1 viruses. A panel of recombinant viruses with their NA gene derived from the A(H1N1)pdm09 A/California/04/09 (CA04), seasonal H1N1 A/New Caledonia/20/1999 (NewCal) or A/Brisbane/59/2007 (Brisbane) was generated in the genetic background of CA04. The H275Y mutation in all three viruses led to a reduced affinity for 3’-sialylactose (3’SL) or 6’-sialylactose (6’SL). Similarly, lowered enzyme activity was observed across H275Y-carrying viruses in 3’SL and 6’SL, with the exception of RG-CA04NA-H275Y at catalyzing 3’SL. Differential 3’SL and 6’SL substrate usage was observed between the NA of seasonal H1N1 and A(H1N1)pdm09 viruses. Reduced infectivity was also observed for recombinant CA04 viruses carrying the H275Y mutation with decreased infectivity in mucin-secreting primary human airway epithelial cells when compared to their oseltamivir-sensitive counterparts. In the ferret model, the pathogenicity of RG-CA04NA-H275Y and RG-CA04BrisbaneNA-H275Y viruses was attenuated albeit the transmissibility was minimally affected when compared to RG-CA04 wild-type virus. In parallel, recombinant seasonal H1N1 viruses encoding the surface glycoproteins of NewCal and Brisbane were tested in ferrets. Results indicated that NewCal and Brisbane viruses carrying the H275Y mutation displayed comparable transmission efficiencies to the wild-type NewCal virus via direct-contact and respiratory-droplet settings. These results suggest that the H275Y NA mutation only leads to a minor reduction in viral fitness, with its transmission potential being minimally affected in the naïve ferret model. / published_or_final_version / Public Health / Master / Master of Philosophy

The seasonal Influenza A viruses are respiratory pathogens causing epidemics annually with mild illnesses, while sporadically, novel influenza viruses emerge and trigger pandemics associated with more widespread and sometimes severe disease. The biological basis for severity of influenza disease remains unclear though it is recognized that the interplay between the influenza viruses and the host immune responses both contribute to viral pathogenesis. As macrophages are key sentinels of the innate immune response and play a crucial role in being the “first responders” as well as contributing to shaping the subsequent (pathogen‐specific) adaptive immune response, the objective of this research was to bring insights on the outcomes of the interactions of influenza viruses with the macrophages. The occurrence of Antibody‐Dependent Enhancement (ADE) of Influenza infection in macrophages was investigated. ADE occurs when non‐neutralized virus‐antibody complexes find alternative entry routes into host cells, mainly through the Fc‐receptor pathway and has been demonstrated predominantly in macrophages. Addition of human serum from some individuals to influenza A virus (either H5 pseudoparticles or pandemic (H1N1) virus) led to enhanced infection of murine macrophage‐like cells as illustrated by a two to five fold increase in detection of influenza M‐gene copies. Immunofluorescence microscopy indicated that serum‐mediated pandemic (H1N1) infection led to an increase in the number of infected cells than in controls. As the fold change in viral gene copies paralleled the fold increase of infected cells I concluded that ADE infection provide pandemic (H1N1) virus with increased opportunity to infect cells rather than simply increase the viral load per cell. In order to strengthen our results, and make them more physiologically relevant, experiments were then performed with human primary cells with clinical sera. However, ADE was not demonstrated in primary human macrophages, suggesting that ADE may be cell type or host specific. The second research question investigated was whether the different state of human primary macrophage differentiation or activation in vitro determined the susceptibility to influenza infection. Recently, work by others has shown a diverse range of macrophage phenotypes that arise by differences in macrophage differentiation and activation. In addition to the classical activation pathway (caMΦ), new mechanisms of activation, designated as alternative activation (aaMΦ), have been reported. Classically and alternatively activated macrophages display different phenotypes and properties, such as molecule expression patterns, cytokine secretion, and gene signatures. This study constitutes the first systematic comparison of Influenza A virus infection of these different subsets of human primary monocyte‐derived macrophages. When assessed for their permissiveness to different influenza A viruses, aaMΦΦshowed greater susceptibility to influenza A infection than caMΦ. This work also documents the receptor patterns and the gene expression profile of these macrophages in response to influenza virus infection in vitro. The results point to differences in susceptibility of the classically and alternatively activated human macrophages to pandemic H1N1 and other influenza A viruses and reveal intrinsic differences between these macrophage subtypes. Further investigations are needed to define the cellular and molecular determinants that define susceptibility of different macrophage subsets to influenza A infection. / published_or_final_version / Public Health / Doctoral / Doctor of Philosophy

Influenza A virus infection is a major cause of human morbidity and mortality. T cell immunity is believed to play critical roles for host defenses against influenza A infection. Once intracellular influenza A infection is established, viral clearance is mainly dependent on virus-specific CD8+ T cells. CD4+ T cells are important for adaptive immunity to natural influenza A infection or vaccination by providing help to B cells for antibody production and also providing help to CD8+ T cells for the generation of cytotoxicity. In addition, virusspecific CD4+ and CD8+ T cells are rich sources of effector cytokines, such as IFN-and TNF-, which can promote the function of antigen presenting cells and have direct antiviral activity. Cross-subtype reactive CD4+ and CD8+ memory T cells also affect the clearance of virus infection even in those who lack virus-specific antibodies. Therefore, the aim of our study is to assess the influenza virus-specific T cell responses and define their possible protective role in pandemic H1N1 virus and seasonal influenza infection in human. First we determined whether healthy adults have the cross-reactivity of memory CD4+ and CD8+ T cells against pandemic virus. In April of 2009, 7 pandemic H1N1 infected patients and 17 their healthy contacts who had no pandemic influenza infection were recruited in this study. By using intracellular IFN-staining and flow cytometry, we examined their pandemic H1N1 virus and seasonal influenza H1N1-specific CD4+ and CD8+ T cell responses. Healthy contacts did have measurable but low frequencies of cross-reactive influenza-specific CD4+ and CD8+ T cells, though the frequencies of these T cells specific to pandemic H1N1 virus were slightly lower than that specific to seasonal H1N1 virus. Furthermore, when compared the pandemic H1N1-specific T cell responses between healthy contacts and patients with pandemic H1N1 infection, we can found that the healthy contacts have higher pandemic H1N1 specific-T cell responses than patients, suggesting these pre-existing pandemic H1N1 specific-T cells may have protection from pandemic influenza virus infection. In addition, we conducted a prospective T cell immunity and influenza surveillance study in a cohort of more than 200 healthy volunteers before the influenza season and investigated whether the pre-existing T cell immunity is related to the protection from influenza infection in the next coming influenza season. Using intracellular IFN-staining assay, we examined their pre-existing seasonal influenza H1N1, H3N2, seasonal influenza B virus-specific CD4+ and CD8+ T cell responses. Due to the small number of cases of influenza infection in the coming influenza season, the results only showed a trend that the subjects who have higher frequency of influenza virus strain-specific T cells may have lower chance to suffer from same strain of influenza infection, which to some extent, reflect the pre-exist memory T cells have association with the protection in the coming influenza season. In conclusion, T cells play an important role in defensing against influenza infection. The higher influenza virus specific-T cells response activity in healthy adults may have a protection against influenza virus infection. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Influenza A virus is one of the most common human pathogens which caused considerable disease burdens through annual epidemics and occasional pandemics. The consequences vary from mild to severe or even fatal. What are the host and viral elements which determine the consequence of infection? In the past 15 years, several avian influenza A viruses including H5N1, H9N2, H7N7 and H7N9 subtypes were found to cross host barrier and infect humans. Question about how avian influenza A viruses gained the ability to replicate in human cells remains unanswered. Studies on host factors associated with virus replication would provide important information for understanding host restriction, virus pathogenesis and for antiviral drug development. Nuclear factor 90 (NF90) is a host protein identified in our previous study to inhibit influenza A virus replication. Antiviral activity of NF90 was also found for other viruses. However, detailed mechanisms for the antiviral function of NF90 remains largely unknown. This study is focused on NF90’s antiviral functions through exploring its relationship with PKR activation and stress granules formation using influenza A virus as a model. I characterized the interaction between NF90 and PKR, and showed the C-terminal of NF90 interacts with PKR in an RNA-binding dependent manner. Using transient and stable NF90 knockdown cells, I found that NF90 is required for PKR activation upon stimulation by dsRNA or infection with a NS1 mutated virus. PKR activation leads to the formation of stress granules and stall of protein translation. I found that NF90 is a core component of stress granules, which may underlie the mechanism for the antiviral activity of NF90. However, NF90 may also complete with PKR for RNA binding and regulate PKR activation. To further delineate the interaction between NF90 and PKR by using influenza A virus, my study constructed a panel of NS1 mutant viruses which were attenuated in antagonizing specific host antiviral pathways. I characterized the NS1 123-127 mutant virus which is unable to inhibit PKR phosphorylation but retained other functions unaffected. It was demonstrated that NF90 mediates PKR-dependent antiviral pathway since NS1 123-127 mutant virus replicated to a comparable level as wild type virus in the NF90 knockdown but not scramble knockdown 293T cells or in the interferon deficient Vero cells. This study for the first time found NF90 serves as a regulator of PKR antiviral pathway. To understand the mechanism for NF90 inhibition of influenza A virus replication, I found that NP, but not the other polymerase subunits, of influenza A virus was targeted to the stress granules. Since NF90 interacts with NP, it is reasonable to postulate that NF90 mediates the localization of NP, and possibly viral mRNA, to the stress granules in order to inhibit influenza A virus replication through regulation of proteins synthesis. In summary, my study provided comprehensive evidence to support a novel NF90-PKR antiviral pathway and suggests that NF90 may play critical roles to balance PKR phosphorylation in response to virus infection in cells. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

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